1. Field of the Invention
This invention relates to a nucleic acid encoding a functional module or domain of a particular peptidoglycan hydrolase, i.e., the phi11 endolysin, a protein which specifically attacks the peptidoglycan cell wall of untreated S. aureus and coagulase negative staphylococci (S. chronogenes, S. epidermis, S. hyicus, S. simulans, S. warneri, and S. xylocus). The phi11 endolysin is active over a broad range of physiological conditions including the pH and calcium concentration of bovine milk. The invention further relates to methods of treating diseases caused by the bacteria for which the phi11 endolysin is specific.
2. Description of the Relevant Art
Bovine mastitis, an infection of the mammary gland, exists on every dairy farm, often with one third of the animals affected. This world wide problem costs two billion dollars annually to the USA dairy industry (Sordillo et al. 2002. J. Mammary Gland Biol. Neoplasia 7 (2): 135-146). Coagulase-negative staphylococci (CNS) and Staphylococcus aureus are major mastitis pathogens in the United States; they were determined to be responsible for 22% and 18%, respectively, of the mastitis cases in a study of New York and Pennsylvania dairy herds (Wilson et al. 1997. J. Dairy Sci. 80: 2592-2598). Antibiotics are the standard treatment for mastitis, but such treatment is often less than 50% effective, a situation which, at times, results in premature culling (Deluyker et al. 2005. J. Dairy Sci. 88: 604-614).
Antibiotic resistance in mastitis treatment is of concern because mastitis is the single most common reason for antimicrobial use in dairy herds (Erskine et al. 2002. J. Dairy Sci. 85 (5): 1111-1118). In a broad study of nine European countries, the USA, and Zimbabwe, 57% of 811 S. aureus isolates from bovine mastitis showed resistance to penicillin (De Oliveira et al., 2001. J. Dairy Sci. 83(4): 855-862). Likewise, 44% of the mastitis strains identified in an Ohio study demonstrated resistance to at least one antibiotic (Rajala-Schultz et al. 2004. Vet. Microbiol. 102 (1-2): 33-42). The Food and Drug Administration (FDA), the United States Department of Agriculture (USDA) and the Center for Disease Control (CDC) promote the development of antimicrobials that reduce risk of resistance development (CDC Action Plan: http://www.cdc.gov/drugresistance/actionplan/html/product.htm). The use of pathogen-specific antimicrobials is expected to reduce the incidence of resistance development (Nathan, C. 2004. Nature 431: 899-902; Walsh, C. 2003. Nat. Rev. Microbiol. 1 (1): 65-70).
To reduce the use of broad range antibiotics and thus decrease the chance of antibiotic resistance development, our goal is to develop pathogen-specific agents that are effective for the treatment of mastitis and as well as for the treatment of clinical multidrug-resistant bacteria, in particular staphylococci, that have developed resistance to antimicrobial drugs. Methicillin/oxacillin-resistant S. aureus is an example of such multi-drug resistant staphylococci. Thus, the invention relates to the protein phi11 endolysin which is specific for and has exolytic activity (i.e., degrades the peptidoglycan of the bacterial cell when exposed externally resulting in lysis of the cell) toward untreated S. aureus and coagulase-negative staphylococci. Further, the invention relates to methods of treating diseases caused by the bacteria for which phi11 endolysin is specific. An additional goal is to express the gene encoding phi11 endolysin and truncated endolysin molecules in mammary glands of transgenic cattle (Donovan at al. 2005. Transgenic Res. 14 (5): 563-567; Wall et al. 2005. Nat. Biotechnol. 23: 445-451). Therefore, both approaches, a strategy of developing transgenic cattle comprising nucleic acid molecules encoding complete and truncated phi11 endolysin and a strategy focused on treatments with phi11 endolysin polypeptide compositions, are important tools for clinical success.
Bacteriophage endolysins specifically degrade the peptidoglycan (PG) of their host cell wall, thus lysing the bacteria, and allowing infective phage to escape. Each phage infects a single cell, replicates itself within the host cell, and then lyses the host cell releasing the progeny phage which then go on to repeat the cycle. Each phage genome codes for lytic proteins that degrade the bacterial cell wall peptidoglycan and allow the newly replicated phage to escape. Although peptidoglycan structure is similar between species, with the oligosaccharide backbone present in all, there is also a great deal of cell wall diversity between different bacterial species. Bacteriophage endolysins are of interest as antimicrobials against Gram-positive organisms (Loessner, M. J. 2005. Curr. Opin. Microbiol. 8 (4): 480-487) due to their high host-specificity and to reports that Gram-positive bacteria are highly unlikely to develop resistance to the peptidoglycan hydrolyzing action of their bacteriophage endolysins (Loeffler at al. 2001. Science 294: 2170-2172; Schuch et al. 2002. Nature 418: 884-889). Peptidoglycan hydrolases have been proposed for human antimicrobial applications (Schuch et al., supra; Fischetti, V. A. 2003. Ann. N.Y. Acad. Sci. 987: 207-214; Fischetti, V. A. 2005. Trends Microbiol. 13: 491-496), and have demonstrated efficacy in mouse models of human disease (Cheng et al. 2005. Antimicrob. Agents Chemother. 49: 111-117; Jado et al. 2003. J. Antimicrob. Chemother. 52 (6): 967-973; Nelson et al. 2001. Proc. Natl. Acad. Sci. U.S.A. 98 (7): 4107-4112), as well as in transgenic murine (Kerr et al. 2001. Nat. Biotech. 19: 66-70) and bovine (Wall et al., supra) mammary glands.
There are at least three peptidoglycan hydrolase activities which have been identified in endolysins: glycosidase, amidase and endopeptidase, (Lopez and Garcia. 2004. FEMS Microbiol. Rev. 28: 553-580). They are encoded by highly conserved domains; each cleaves a unique peptidoglycan bond (Loessner, supra). The conserved domains are ˜200 amino acids in size (Huard et al. 2003. Microbiology 149 (Pt 3): 695-705; Rigden et al. 2003. Trends Biochem. Sci. 28: 230-234; Bateman and Rawlings. 2003. Trends Biochem. Sci. 28: 234-237), and are readily identified using common domain databases (Uniprot: http://www.pir.uniprot.org/index.shtml; Pfam: http://www.sange.ac.uk/cgi-bin/Pfam/getacc?PF04650; SMART: http://smart.embl-heidelberg.de/, or NCBI conserved domain data base). The domains are referred to as modules because they can often function independently of the remainder of the native endolysin (Navarre et al. 1999. J. Biol. Chem. 274 (22): 15847-15856; Yokoi et al. 2005. Gene 351: 97-108; Morita et al. 2001. FEBS Lett. 500: 56-59; Donovan et al. 2006b. Appl. Environ. Microbiol. 72: 5108-5112), and maintain activity when fused to create novel, recombinant, fusion hydrolases (Diaz at al. 1990. Proc. Natl. Acad. Sci. USA 87:8125-8129; Garcia at al. 1990. Gene 86: 81-88; Donovan et al. 2006 a. Appl. Environ. Microbiol. 72: 2988-2996).
In the quest for antimicrobials against multidrug-resistant staphylococci including, for example, mastitis-causing bacteria and methicillin/oxacillin-resistant S. aureus, agents must be found that can target our pest organisms very specifically. This is not just to reduce the potential for resistance development, but also to prevent damage to commercially important organisms that are necessary for the downstream processing of milk into yogurt and cheese. The prevention of infection and disease caused by multidrug-resistant organisms, including mastitis, would not just benefit animal health, and food quality, but also human health. Any antimicrobial that is specific for a given pathogen will potentially reduce the use of broad range antibiotics and thus also help prevent the onslaught of multi drug resistant varieties.
Thus, to counter the rise of drug resistant pathogenic bacteria, there is a need for new specific antimicrobial treatments. Reagents shown to be very specific for the genera, species or substrains of concern would give better effective control of economically important diseases and therefore are ideal candidates for therapeutic treatments.